Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. Carbohydrates are the main respiratory substrates for plants, furnishing the malate and the acetyl-CoA necessary for the functioning of the Krebs cycle. Fifteen years ago it was still considered that, during the day, photosynthesis allowed starch synthesis in such amounts that the plant, particularly the root system, was never deprived of sugars. However, it is now known that carbohydrate starvation is common in most higher plants. Indeed, microbial, insect, or herbivore attacks or reduction in light intensity or temperature may cause a substantial decrease in photosynthesis, and thus lead to starvation.Since the end of the seventies, carbohydrate starvation has been studied in a number of plant species: wheat (28, 29), maize (14, 18), barley (8), pearl millet (1), pea (20,26,27), soybean (13, 25), sycamore (7, 10, 12, 17), etc. These studies have shown that in most cases, sugar starvation triggers the following sequence in plant cells: (a) the depletion of intracellular carbohydrate content and the subsequent decrease of respiration (1,12,18,20,25); (b) the breakdown of lipids and proteins (1,7,12,28) and a decline in the respiratory quotient from 1 to 0.75 (18); (c) an increase in inorganic phosphate ( 12,17), phosphorylcholine (7, 17), and free amino acids (10), and a concomitant decline in nucleotides (17, 18) and glycolytic enzymatic activities (12); and (d) the more or less marked disappearance of some cell ultrastructures (1, 29).The origin of the respiratory decrease during starvation was first attributed to carbohydrate depletion, by way of limitation of the substrate either for respiration or for biosynthetic processes; however, some experiments showed that root respiration rate was not a simple function ofcarbohydrate supply (8). Journet and co-workers (12) reported that during starvation the decrease in uncoupled respiration of sycamore cells was attributable to a progressive decrease in the number of mitochondria per cell; these authors concluded that the availability of respiratory substrates for the mitochondria does not determine the respiration rate of starved cells.In the present work, we investigated changes in 02 consumption, different organic compounds (sugars, fatty acids, proteins, adenine nucleotides), enzyme activities, and physical parameters (fresh and dry weight, osmolarity) in excised maize (Zea mays) root tips from the beginning of glucose starvation to tissue dea...

Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).

Oxygen uptake and energy charge were monitored during aging of excised maize root tips and related to the soluble sugar content and exogenous sugar supply.Oxygen uptake declined imsediately after excision to 50 to 30% of its initial value after 8 and 24 hours of aging at 25 C. There was also a sharp decline of the total sugar content (glucose, fructose, and sucrose). Starch content was very low at the time of excision and aimost neglgible 5 hours later. During the same period, the respiratory quotient declined from I to 0.75 and then remained stable.The addition of exogenous sugars induced a rapid rise of the respiratory rate which stabilized at a level correlated to the external sugar concentration. Addition of 0.2 molar glucose was necessary to restore the respiratory rate to the initial, also the maximum, level. These results indicate that metabolic activity of root tips is hily reliant on sugar import and carbohydrate reserves at the time of excision cannot compensate for the cessation of import. The control of respiration by substrate supply is in good agreement with the failure for dinitrophenol to stimulate oxygen uptake in aged sugar-depleted root tips.The energy charge remained constant at about 0.9, irrespective of the presence or absence of glucose and in spite of a large decline of respiratory activity in aged, sugar-depleted tissues.In normoxia as defined by Pradet and Bomsel (16), the respiratory rate of tissues is not limited by the 02 partial pressure.Under such conditions, most of the biological energy provided to the cells comes from sugar oxidation through respiratory pathways. The sugar supply of non-chlorophyllous tissues varies depending upon factors which affect the efficiency ofcarbon fixation by leaves and transport of the recently synthesized carbohydrates (5, 7, 13, 21).The question arises as to how metabolism adjusts to such fluctuation. Stimulation of root respiratory rate by light after darkness has been reported previously (6, 9). Hatrick and Bowling (10), using sunflower and barley, reported evidence for a complete dependence of root respiration on the rate of assimilate translocation from the shoot, suggesting that roots of young herbaceous plants have no reserves which might reduce the effect of fluctuations in the rate of translocation of photosynthetic sugars.The present study is part of an effort to understand the factors which limit and control the metabolic activity of roots. Our approach was to relate 02 uptake, taken as a measure of the metabolic activity, to soluble sugar and adenine nucleotide content of the tissues at various times after excision. The data reported demonstrate the influence of exogenous sugars on the metabolic activity of root tips. The significance ofenergy charge as parameter of normoxic cellular metabolism is discussed. MATERIALS AND METHODSPrimary root tips, 0.5 cm, were cut from maize seedlings (Zea mays L. INRA 402) germinated for 3 days at 25 C between sheets of filter paper soaked with 2 mm CaCl2.Determination of Gas Exchange. Measureme...

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same "synthetic family" showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90-100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the β-cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2-4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30-35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30-35 to 90-100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.

ABSTRACFYoung intact plants of maize (Zea mays L cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretratment) for 18 hours before excision of the 5 millimeter root apex and treatment with sictly anaerobic conditions (anoxia). Hypoxic accUmation pve rise to lrer amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips trferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was assoted with incsed activity of alcohol dehydrogenase (ADH), and indction of ADH-2 isozymes. Roots of Adbl-mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of N03-. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NOi-to NO2-. These tips (9-11, 25) is strongly suggestive of such acclimation, but the 02 concentrations to which cells were exposed during published experiments often were not controlled or defined closely, and were rarely, if ever, strictly anaerobic. Anaerobic polypeptides were induced by hypoxia in roots of rice (PO2 < 5 kPa) and wheat (PO2 < 2 kPa) continuously maintained at different constant concentrations of 02 (3, 4). However, the significance of these proteins, which include ADH2 and some of the glycolytic enzymes was not examined and remains unclear.Nitrate ion may play a special role during anoxia, by acting possibly as a terminal electron acceptor in respiration in the absence of molecular 02, with NADH-dependent reduction of nitrate to nitrite via NR in 'nitrate respiration' (7,12,18). It's significance lies in the regeneration of NAD+, essential for the continuation of glycolysis. Additionally, it has been suggested that competition for cytoplasmic pools of NADH between NR and ADH can occur, thereby lowering the rate of ethanol production (7,12 Anaerobic Treatment. Root apices, 5 mm long, were excised and placed in groups of 10 in 10 ml-volume glass vials with rubber puncture caps in 2.0 ml of the solution in which the roots had been pretreated, but supplemented with 100 mm glucose.Hypodermic needles through the rubber caps were used to gas the solution and headspace, one needle bubbling gas into the solution, the other to give gas exit from the vial. Equilibrium between these small volumes and a new gaseous atmosphere was attained in a few minutes. Roots from the fully aerobic and the hypoxic pretreatments were gassed either with 40 to 50% (v/v) 02 in N2, or with O2-free N2 (the anaerobic treatment, N2 = 99.99%). There was 3-fold replication.Extraction and Estimation of Adenine Nucleotides. Solution was forced out from each vial by the gas mixture with...

The effect of 02 partial pressure on the germination and the respiration of 12 cultivated species was studied. The reciprocal of the time necessary to observe rootlet emergence in 50% of the seeds was used to approach the germination rate. The maximum germination and respiration rates were reached in most seeds at 02 pressures close to that ofair. Decreasing the 02 pressure produced a gradual decrease of the germination rate. The seeds could be classed in two groups according to their response to IOW 02 pressures. Group I includes lettuce, sunflower, radish, turnip, cabbage, flax, and soybean: at 02 pressures close to 2 kilopascals, the germination in this group was stopped and the adenylate energy charge was lower than 0.6. Group II includes rice, wheat, maize, sorghum, and pea. The germination rate of these seeds was also gradually decreased by lowering the 02 partial pressure but germination still occured, very slowly, at 0.1 kilopascal; the adenylate energy charge remained higher than 0.6. These differences in the germination rates and adenylate energy charge values could not be explained by differences in the sensitivity of respiration to 02-The effect of P02' on seed germination has been studied by only a few authors (9,15,17,28). In two graminae, rice and Echinochloa crus galli, the coleoptile is able to grow under anaerobiosis (10,26,30) and the coleorhiza of Echinochloa is able to emerge (29). Apart from these two exceptions, seed germination, as well as other growth processes in higher plants, requires 02-The respiration of seeds increases dramatically during the first hours of imbibition, which corresponds to germination phase I ( 11); afterwards, it stabilizes, or increases more slowly (germination phase II), until rootlet protrusion which completes the germination process (5). Heichel and Day (9) observed that the germination of monocots occurs at P02 as low as 2 kPa, whereas the germination of most dicots required higher P02. They concluded that oxidation systems involved in germination require 02 levels greater than 2 kPa and that these systems differ in monocots and dicots. The Cyt oxidase is the site of most respiratory oxidations (12,19 about 15 kPa (18). This has been explained by the presence of barriers to the diffusion of 02 from the surface of the seed envelopes, to the internal membrane ofthe mitochondria (7,20). Thus, it could be hypothesized that those seeds which are unable to grow below 2 kPa O2 require higher P02 for respiration than the other seeds.Oxidative phosphorylation is not the only mechanism that produces ATP in seeds. It has been shown that fermentation is very active during the initial phases ofgermination ofsome seeds (14). Nevertheless, it was shown recently that fermentation contributes very little to ATP regeneration in lettuce seeds (24). This observation led to a second hypothesis (1): the seeds which are unable to germinate at P02 lower than 2 kPa regenerate less ATP by fermentation than the others.It has been established that the value of the ATP/ADP ratio,...

Energy charge, adenine nucleotide levels, and protein synthesis were studied during the transfer of rice seedlngs from air to anoxia. Within minutes, the energy charge value dropped from 0.90 in air to 0.50 in the seed and 0.60 in the coleoptile after the transfer to a nitrogen atmosphere, and then increased to a value of 0.80 during the subsequent hours. The sum of nucleotides also dropped to 60% of the value in air in the seeds and to 30% in the coleoptiles. However, during the anaerobic growth of coleoptiles, a considerable increase in the nucleotide pool occurred.The incorporation of amino acids into proteins was measured at different stages in anoxic treatment. In rice embryo, we observed a considerable protein synthesis correlated with a high value of energy charge under anoxia. The analysis oflabeled proteins by two-dimensional polyacrylamide gel electrophoresis showed a modified pattern of polypeptides synthesized during anoxic treatment. Some of these proteins were intensively labeled and appeared to be induced by anaerobic treatment.Our data indicate that high metabolic activity occurs in rice embryo under anoxia, which can be correlated with a high energy charge value. These phenomena may be part of the mechanisms which permit the adaptation of rice embryos to anaerobiosis.In nonchlorophyllous plant tissues and in animals, it is generally assumed that ATP synthesis is drastically reduced when oxidative phosphorylation is inhibited by a lack of 02. The knowledge of the balance between ATP regeneration and utilization for metabolic work should permit a better understanding of the mechanisms used by these organisms to survive under anoxic conditions.In animal tissues, there is a rapid inhibition of protein synthesis after a few min of anoxia (10,12 Preliminary data obtained in our laboratory indicate that rice embryos or coleoptiles germinated in anoxia or transferred from air to nitrogen have a high energy charge (20, 21) and an active RNA (2) and DNA synthesis (16).The purpose of this paper is to study the effect of anoxia on adenine nucleotide levels, energy charge, and incorporation of labeled amino acids into proteins in rice embryos. The correlation between energy charge and protein synthesis in anoxia is discussed, and anoxic protein patterns sustain the hypothesis of an adaptation of rice embryo to anaerobic conditions. MATERIALS AND METHODSPlant Materials. Rice seeds (Oryza sativa L., var. Cigalon) cultivated by the Station d'Amelioration des Plantes (INRA, Montpellier, France) were mechanically husked and sterilized with commercial (NaOCl (150 g Cl/l), as described previously (16).Germination of Rice Seeds. Seeds were placed in glass flasks with water and shaken at 26 C in darkness. They remained under aerobic conditions for 40 to 48 h until coleoptiles reached 4 to 5 mm.For anoxia, samples were placed in a nitrogen flow (100 ml/ min), as previously described (16). Oxygen content was checked with an 02 analyzer (WOM, Mecanalyse, France) or by gas chromatography. PO2 in N2 was lower than...

The respiration and fermentation rates were compared in germinating seeds of 12 different cultivated species from five families. In air, fermentation contributes significantly to the energy metabolism only in some species (pea, maize), but is generally negligible when compared to respiration. The fermentation rate under anoxia was related either to the metabolic activity under air or to the adenine nucleotide content of the seeds: it was generally higher in seeds which contain starchy reserves (rice, maize, sorghum, pea), than in seeds which do not contain starch (lettuce, sunflower, radish, turnip, cabbage, flax); however, it was similar in wheat, sorghum (starchy seeds), and soya (nonstarchy seeds). The value of the energy charge of all the seeds was lower under anoxia than in air: after 24 hours under anoxia, it was higher than 0.5 in the starchy seeds and in soya and it was around 0.25 in the other fatty seeds.anaerobiosis in seed germination.The present work is part of a study in which we used a comparative, physiological approach with seeds of 12 different species belonging to five families. When studying the effect of the P02 on the rate of germination (2), we found that the seeds could be classed in two groups: group I included fatty seeds; in these seeds, radicle emergence did not occur below I kPa 02. Group II included starchy species, which were able to germinate at 02 partial pressures below 0.1 kPa. In the present work, the rate of 02 uptake, the rate of ethanol and lactate synthesis, and the value of AdN ratios (ATP/ADP or AEC), were used as indicators of ATP regeneration.We found that, in aerated conditions, respiration was the dominating ATP-regenerating pathway in all seeds, whereas fermentation contributed significantly only in some species. Starchy seeds appeared to maintain a higher energy metabolism under anoxia than the fatty seeds, although the two groups overlap to some extent.When dry seeds are imbibed, the respiratory activity, characterized by 02 uptake and CO2 evolution, starts extremely rapidly (5). For a long time, it was thought that this oxidative process was not coupled to ATP production and some original mechanisms were recently proposed (17) to explain the origin of the ATP needed for the biosynthetic work done during germination. Even under well-aerated conditions, ethanol and lactate accumulation may occur. It is generally considered that, during the first hours of imbibition, seeds are under 'natural anaerobiosis' (15). However, oxidative phosphorylation was demonstrated in mitochondria of the freshly imbibed Arachis hypogea axis (28), and, using an in vivo method, in lettuce seeds after 15 min of imbibition (12). Hence, the respiration of seeds is probably an important source ofATP during the initial phases ofgermination (19).In a recent study, we found that the fermentative pathways contributed very little to ATP regeneration in germinating lettuce seeds (21). Moreover, their respiratory activity is saturated by PO2' below that of air (2,22). Such characteristics a...